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In JoVE (1)
- गतिशील लाइव सेल इमेजिंग के लिए मेजबान पैथोजन सहभागिता के अध्ययन के लिए एक ऑप्टिकल जाल का प्रयोग करें
Other Publications (7)
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Articles by Carlos E. Castro in JoVE
JoVE Immunology and Infection
गतिशील लाइव सेल इमेजिंग के लिए मेजबान पैथोजन सहभागिता के अध्ययन के लिए एक ऑप्टिकल जाल का प्रयोग करें
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University
एक विधि के लिए व्यक्तिगत का चयन करें, हेरफेर वर्णित है, और छवि रहते रोगज़नक़ों एक कताई डिस्क खुर्दबीन एक ऑप्टिकल मिलकर जाल का उपयोग कर. ऑप्टिकल जाल स्थानिक और लौकिक जीवों के नियंत्रण प्रदान करता है और उन्हें मेजबान कोशिकाओं के लिए आसन्न स्थानों. प्रतिदीप्ति माइक्रोस्कोपी कोशिकाओं को कम से कम गड़बड़ी के साथ गतिशील कहनेवाला बातचीत कब्जा है.
Other articles by Carlos E. Castro on PubMed
Caspofungin Therapy of Neonates with Invasive Candidiasis
Invasive candidiasis is an increasing problem in neonatal intensive care units worldwide and is an important cause of morbidity, mortality and prolongation of hospital stay. Despite administration of amphotericin B, invasive candidiasis in neonates is sometimes complicated by persistent fungemia and refractory invasive candidiasis. The problem has been augmented by the increasing prevalence of non-albicans species that often are resistant to fluconazole and to amphotericin B.
Vascular Access-related Infections in HIV Patients Undergoing Hemodialysis: Case Description and Literature Review
Poor immune status, the use of a vascular access different from an AV fistula, and intravenous drug use (IDU) may favor increased rates of vascular access infections among HIV infected patients on hemodialysis. Staphylococcus spp. and Streptococcus spp. are the main cause of these infections, but Gram-negative rods and fungi have been found as well. Using an AV fistula when possible, and eliciting a history of IVDU on every visit may prevent this type of infection. When infections are present, coverage for both Gram-positive and negative organisms is recommended. Additional studies specifically addressing the issue of vascular access infection in HIV infected patients are required.
The Alphabeta T Cell Receptor is an Anisotropic Mechanosensor
Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric alphabeta, CD3 epsilon gamma, CD3 epsilon delta, and CD3 zeta zeta subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3 epsilon lobe that they approach diagonally, adjacent to the lever-like C beta FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3 epsilon and CD3 gamma in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force ( approximately 50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.
Optical Trapping with High Forces Reveals Unexpected Behaviors of Prion Fibrils
Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and extend fibril length.
Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions
The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.
DNA Origami-based Nanoribbons: Assembly, Length Distribution, and Twist
A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.
Physical Properties of Polymorphic Yeast Prion Amyloid Fibers
Amyloid fibers play important roles in many human diseases and natural biological processes and have immense potential as novel nanomaterials. We explore the physical properties of polymorphic amyloid fibers formed by yeast prion protein Sup35. Amyloid fibers that conferred distinct prion phenotypes ([PSI(+)]), strong (S) versus weak (W) nonsense suppression, displayed different physical properties. Both S[PSI(+)] and W[PSI(+)] fibers contained structural inhomogeneities, specifically local regions of static curvature in S[PSI(+)] fibers and kinks and self-cross-linking in W[PSI(+)] fibers. Force-extension experiments with optical tweezers revealed persistence lengths of 1.5 μm and 3.3 μm and axial stiffness of 5600 pN and 9100 pN for S[PSI(+)] and W[PSI(+)] fibers, respectively. Thermal fluctuation analysis confirmed the twofold difference in persistence length between S[PSI(+)] and W[PSI(+)] fibers and revealed a torsional stiffness of kinks and cross-links of ~100-200 pN·nm/rad.
